Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli

被引:39
作者
Dai, MH
Ziesman, S
Ratcliffe, T
Gill, RT
Copley, SD [1 ]
机构
[1] Univ Colorado, Dept Mol Cellular & Dev Biol, Boulder, CO 80309 USA
[2] Univ Colorado, Cooperat Inst Res Environm Sci, Boulder, CO 80309 USA
[3] Univ Colorado, Dept Chem & Biol Engn, Boulder, CO 80309 USA
关键词
D O I
10.1016/j.ymben.2004.09.002
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05-0.7 % based on the number of protoplasts subjected to fusion. This frequency is three-four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E coli and Providencia alcalifaciens. (c) 2004 Elsevier Inc. All rights reserved.
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页码:45 / 52
页数:8
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